Formation of the catecholamine release-inhibitory peptide catestatin from chromogranin A - Determination of proteolytic cleavage sites in hormone storage granules

The catestatin fragment of chromogranin A is an inhibitor of catecholamine release, but its occurrence in vivo has not yet been verified, nor have its precise cleavage sites been established. Here we found extensive processin g of catestatin in chromogranin A, as judged by catestatin radioimmunoassay of size-fractionated chromaffin granules. On mass spectrometry, a major ca testatin form was bovine chromogranin A(332-364); identity of the peptide w as confirmed by diagnostic Met(346) oxidation. Further analysis revealed tw o additional forms: bovine chromogranin A(332-364) and A(343-362). Syntheti c longer (chromogranin A(332-364)) and shorter (chromogranin A(344-364)) ve rsions of catestatin each inhibited catecholamine release from chromaffin c ells, with superior potency for the shorter version (IC50 similar to 2.01 v ersus similar to 0.35 mu M). Radioimmunoassay demonstrated catestatin relea se from the regulated secretory pathway in chromaffin cells. Human catestat in was cleaved in pheochromocytoma chromaffin granules, with the major form , human chromogranin A(340-372), bounded by dibasic sites. We conclude that catestatin is cleaved extensively in vivo, and the peptide is released by exocytosis, In chromaffin granules, the major form of catestatin is cleaved at dibasic sites, while smaller carboxyl-terminal forms also occur, Knowle dge of cleavage sites of catestatin from chromogranin A may provide a usefu l starting point in analysis of the relationship between structure and func tion for this peptide.